Mycobacterium tuberculosis (Mtb) is a significant global health problem, infecting one third of the world's population, and is the leading cause of death from a curable infectious disease. Attributable mortality rates were reported as 1,300,000 in 2012. Despite infection in humans since ancient times, we lack complete understanding of Mtb pathogenesis. Therapy is challenging due to the emergence of drug resistance, and the ability of Mtb to remain latent for extended periods in a state referred to as non-replicating (NR) Most current therapies are ineffective at eradicating NR Mtb, which has the potential to reactivate, causing disease. New therapeutics are needed that target both replicating (R) and NR Mtb. We have identified a novel class of compounds, nitrofuranyl calanolides (NCs), which achieve this goal. NCs are very potent against R and NR Mtb, are selective, and tolerated well by primary human cells. In addition, their ability to kill Mtb in primary human macrophages makes them an important class to study. Our project focuses on using NCs as tools to discover the biology of the dormant NR state and new druggable targets. We will accomplish this by finding NC-resistant bacteria that have mutations in the essential target(s), in addition to using metabolomics to identify essential pathways that are altered by NCs. Our preliminary data show that rv2466c, a gene of unknown function in Mtb, is an activating enzyme for NCs, leading to production of several conversion products in vitro, proposing a nitroreductase function for this enzyme. We will identify and synthesize these conversion products, and test their activity against Mtb. If active, these conversion products will be also used to identify potential targets important in killing R and NR Mtb. Moreover, we predict that in addition to its role in NC activation, rv2466c also plays a role in Mtb pathogenesis and dormancy. Evidence from transcriptome studies predict it to be important during stress conditions encountered in the host, which is supported by our preliminary findings showing its importance in oxidative stress resistance. We will further evaluate significance of Rv2466c by generating an rv2466c-knockout strain, and evaluating its phenotype in response to physiologic stresses and during in vivo mouse infections. Furthermore, we will evaluate its binding partners using co- immunoprecipitation strategies, while using information from the already available crystal structure to guide us better delineate sites important for enzymatic and binding activity.